Zolpidem, systematic name N,N-dimethyl-2-[6-methyl-2-(4-methylphenyl)imidazo[1,2-a]pyridin-3-yl]acetamide, is a widely prescribed drug designed to counteract sleep and brain disorders, notably insomnia. The drug is classed as a non-benzodiazepine hypnotic and acts on the gabba aminobutyric acid receptors. Upon ingestion, zolpidem is fast-acting and has a half-life of 1.5-3 hours. Metabolism is mediated by cytochrome P450, producing more than 80% 4-[3-(2-N,N-dimethylamino-2-oxoethyl)-6-methylimidazo[1,2-a]pyridin-2-yl]benzoic acid (M1) formed via the corresponding hydroxy metabolite, and a relatively smaller amount of 3-(2-N,N-dimethylamino-2-oxoethyl)-2-(4-methylphenyl)imidazo[1,2-a]pyridin-6-yl carboxylic acid (1-4). Despite its proven therapeutic effectiveness there have been increasing reports of dependence, recreational and criminal abuse (5-7) and of life-threatening side-effects due to hallucinations and impaired driving (8). Villain et al (9) report zolpidem to be ranked first in a list of drugs used in drug-facilitated sexual assault. This potential for adverse events or abuse in individuals taking or with access to zolpidem creates a need in clinical and forensic toxicology for its detection and/or determination using practical and inexpensive analytical methods.
Analytical methods that have been used to detect and determine zolpidem include HPLC, LC-MS-MS, GC and GC-MS. A complex and equipment-intensive, column-switching HPLC method incorporating a clean-up and pre-concentration phase targeted M1 as a marker of overdose as it is the most abundant zolpidem marker in both plasma and urine (1). A GC-MS method requiring sample extraction and compound derivatisation is described for detecting M1 for up to 72 hours in two cases of alleged zolpidem drug-facilitated assault (10). The less costly and practical immunoassay has also been used to detect and quantify zolpidem. The immunoassay has many advantages over other mainstream analytical formats such as GC-MS and LC-MS including cost, ease of use and its amenability for manufacture in a compact, portable format for at the scene use. A radioimmunoassay (RIA) and an EMIT-type assay have been described. The RIA describes an immunogen in which the protein crosslinker is incorporated at the 3-position of the fused heterocycle which results in detection of zolpidem, with no reported cross-reactivity towards the metabolites (11). The RIA is extremely sensitive but has associated health and safety issues. Generally, RIAs have low commercial uptake and the inventors are not aware of a commercially available zolpidem RIA. A commercial kit is available for zolpidem forensic screening in whole blood, serum and urine that has a detection cut-off value of 25 ng/ml (Catalog 233 ELISA zolpidem insert, Immunalysis). The kit was reported not to detect M1 at 1000 ng/ml (8). A follow-on immunoassay developed by the same company for the detection of zolpidem in urine reports a detection limit of 5 ng/ml and a detection window of eight hours in an individual described as an infrequent user; at 16 hours zolpidem could not be detected (12). A commercial ELISA kit produced by International Diagnostic Systems detects zolpidem at 75 ng/ml. None of the kits report detection of M1.
Due to zolpidem's rapid and varied inter-individual metabolism (8, 9, 12), the current immunoassays detecting only the parent molecule are insufficently sensitive to be used reliably as a screening tool for detecting zolpidem mis-use and drug-impaired driving in patient samples beyond approximately 8-24 hours. To overcome this inadequacy, the inventors devised and developed an immunoassay based on detection of zolpidem and its main metabolite M1. The disclosure enables the sensitive immuno-detection and determination of zolpidem and its main metabolite in patient samples, and extends the time period in which zolpidem can be detected following ingestion.
To the extent that the following publications do not conflict with the teachings of the present disclosure, they are incorporated herein by reference:    1. Ascalone V. et al. (1992). J. Chromatogr. 581: 237-250    2. Hempel G. and Blaschke G. (1996). J. Chromatogr. B 675: 131-137    3. von Moltke L. et al. (1999). Br. J. Clin. Pharmacol. 48: 89-97    4. Salva P. and Costa J. (1995). Clin Pharmacokin. 29: 142-153    5. Madea B. and Muβhoff F. (2009). Dtsch. Arztebl. Int. 106:341-347    6. Maravelias C et al. (2009). Am. J. Forensic Med. Pathol. 30: 384-385    7. Victorri-Vigneau C. et al. (2007). Br. J. Clin. Pharmacol. 64: 198-209    8. Reidy L. et al (2008). J. Anal. Tox. 32: 688-694    9. Villain M. et al. (2004). Forensic Sci. Int. 143: 157-161    10. Lewis J. and Vin J. (2007). J. Anal. Tox. 31: 195-199    11. De Clerck I. and Daenens P. (1997). The Analyst 122: 1119-1124    12. Huynh K. et al. SOFT 2009, Oct. 19-23, 2009, Oklahoma, Program and Abstracts